GSM7976849: RPE_dox_gp replicate2; Homo sapiens; RNA-Seq
Cells were collected by trypsinization, washed with PBS after which cells were fixed with 0.5mM dithiobis(succinimidyl propionate)(DSP) in Sodium Phosphate buffered Saine (pH 8.4) for 45 minutes at room temperature at a concentration of 1 million cells per 2.5ml. Next, DSP was neutralized by incubating the cells with quench buffer (100mM Tris, pH 7.5, 150mM NaCl) for 10 minutes and cell clumps were removed usmg a 70µm cell strainer. Cells were icubated for 30 minutes with BP buffer (PFBB:PBS, 1:1, supplemented with 0.1% Triton•X-100 and 2 U/mL RNAsin plus) to permeabilize the cells, after which samples were incubated overnight with antibodies in BP buffer. Samples were filtered using a 40 um cell strainer and incubated with DAPI (0.2 ug/mL) prior to loading of samples on the flow cytometer.  Cells were sorter into the captuture plates on a BD Influx cell sorter. Single Cell Discoveries, where single-cell RNA sequencing was performed according to the SORT-seq protocol. Cells were heat-lysed at 65° C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit